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Bio 370, spring 2011 Biolistic Transformation. page 1 Biolistic "Gene Gun" transient gene expression assay. Purpose: to demonstrate biolistic...

I'm doing an experiment using a gene gun, and I need to answer a question that I'm stuck on. The question asks that starting with a 2.5 microgram/microliter solution of DNA, and 100 mg of M-10 tungsten particles, how would you prepare a series of 5 mg DNA per 1.2 mg particles solutions for transformation? How many total shots would you get from this preparation?

Am I supposed to set up 2.5ug/ul = 5mg/(x)ul and use this answer? I have attached my protocol if more information is necessary. I don't have any information on how to come about with this answer, so I am really very confused. Please help!

Bio 370, spring 2011 Biolistic Transformation. page 1 Biolistic “Gene Gun” transient gene expression assay. 1. Purpose: to demonstrate biolistic transformation and transient expression of a reporter gene as a tool in Developmental Biology 2. Overview: Transient expression is a method for the rapid, temporary introduction of gene expression constructs into specific cells or tissues of an organism. Unlike stable transformation (as with Agrobacterium-mediated transformation), transient expression involves only somatic cells, and does not transform the germ line. Thus, expression of the introduced transgene is only temporary (transient, a few hours to a few days), and is not transmitted to the next generation. The gene gun was developed in 1987 by John Sanford and coworkers at Cornell University. The earliest gene gun used 22 caliber explosive nail gun cartridges to propel DNA coated tungsten microprojectiles into living tissues. The patent was sold to Du Pont in 1990. Newer versions use helium gas to propel microprojectiles made from tungsten, gold, or silver. While often used for plant biology, the gene gun has been used for a number of other organisms, including Paramecia (Dr. Hennessey's lab in this department), Drosophila , C. elegans , fungi, and mammalian (including human) cells. It is capable of both transient expression, and under the right conditions, stable transformation The following figures and description of the instrument, are taken and modified from the instruction manual for the Biolistic PDS-1000/He Particle Delivery System that will be used for this laboratory. This system uses high-pressure helium from a pressurized cylinder to rapidly accelerate microprojectiles into the target cells (in this case, tobacco leaf cells). Figure 1. main unit of PDS-1000 “gene gun” Microcarrier Launch Assembly Power Switch ON/OFF Helium Pressure Gauge Target Shelf Bombardment Chamber Door Vacuum Gauge Vac/Vent/Hold Switch Vacuum/Vent Rate Control Valves Fire Switch Disk Retaining Cap
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Bio 370, spring 2011 Biolistic Transformation. page 2 Components of the main unit of the instrument are shown in Figure 1 . The main unit consist of the bombardment chamber itself, with holders for rupture disks, for the microprojectile (launch assembly), and a shelf for the target samples. The main unit also has three control switches, a vacuum gauge, and a pressure gage. As shown in Figure 2, the instrument also requires a vacuum pump with connection tubing, and components for delivery of high pressure helium to the main unit (helium regulator, pressure regulator, solenoid valve, and connection tubing for delivery of the helium gas). The instrument is often housed under a laminar flow hood for applications that require sterile conditions. Within the main unit of the Biolistic PDS-1000/He system, high pressure helium, released by breakage of a rupture disk, causes the rapid acceleration of a macrocarrier sheet (also called a “flying disk”) that is loaded with millions of microscopic tungsten or gold microprojectile particles toward target cells at very high velocity. The microprojectiles are coated with DNA for transformation (you will do the coating as part of this exercise). Because the particles are so small, the process occurs under partial vacuum to minimize resistance that would occur if the particles were propelled through air. The biolistic processes is summarized in Figure 3 at right. When the “fire” switch is pressed, helium pressure builds up in the Gas Acceleration Tube above the Rupture Disk , until the disk breaks open (the rupture disks are calibrated for breakage at specific pressures; the higher the pressure, the greater the acceleration). This Figure 2. Additional components of PDS-1000 Helium Pressure Regulator Vacuum (reinforced PVC) Tygon Tubing 2.5 ft PEEK Tubing 6 ft PEEK Tubing Tank of Pressurized Helium (user provided) Rupture Disk Retaining Cap 3-Way Helium Metering (solenoid) valve Microcarrier Launch Assembly Helium Regulator Target Plate Shelf Figure 3. The biolistic transformation process Before Gas Acceleration Tube Rupture Disk Macrocarrier Stopping Screen Target Cells DNA-coated Microcarriers After
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